T cell receptors (TCRs) have emerged as a new class of immunological therapeutics. Clinical trials with TCR gene-modified T cells have shown that objective clinical responses can be obtained for patients with advanced malignancies. Similar approaches are in development for treatment of infectious disease. However, there is considerable debate over the nature of the TCR to be used in engineered T cells, and whether naturally occurring TCRs can be improved. Emphasis has been on identifying natural ?high affinity? TCRs, and these have been emphasized in clinical trials. As T cell potency can sometimes be strengthened with the affinity of the TCR for antigen, there have also been efforts to use TCRs engineered for enhanced antigen affinity in immunotherapy. However, adverse events, including deaths, have occurred in some trials with gene-modified T cells. In some cases, this is clearly attributable to TCR cross-reactivity. Moreover, as high affinity can curtail function and low affinity TCRs are clearly functional, the presumption that improved TCR affinity is better for immunotherapy is questionable. In this multi-PI proposal, we propose an ambitious and innovative program to ask and answer how to build better TCRs for immunotherapy. Our overall hypothesis is that structure-guided design coupled with comprehensive in vitro and in vivo functional studies can be used to engineer TCRs for improved antigen recognition while limiting off-target cross-reactivity. To achieve this, we will combine the T cell biology and immunotherapy expertise of the Nishimura lab at Loyola with the TCR structure and biophysics expertise of the Baker lab at Notre Dame. We will also incorporate emerging concepts of ?2D affinity? measurements and assess how they relate to specificity and other biophysical parameters, with 2D measurements to be performed by the Evavold lab at Emory. Using the MART1 tumor antigen as a model and beginning with the clinically relevant DMF5 TCR, we propose the following three aims: 1) Determine how structure-guided manipulations of TCR binding impact antigen recognition and in vivo function; 2) Generate improved TCR variants through iterative cycles of structure-guided design and biophysical/functional characterization, emphasizing the capacity to engineer specificity independently of affinity; 3) Assess the generalities of the lessons learned by applying the results from DMF5 to one or more unrelated MART1-specific TCRs. The completion of the aims will lead to a better understanding of how TCRs recognize antigen and how to most effectively engineer TCRs for optimal function in immunotherapy.